22 October 2008. In new work on their mouse model of 22q11 deletion syndrome, the research groups of Maria Karayiorgou and Joseph Gogos at Columbia University report that the absence of Zdhhc8, a palmitoyltransferase enzyme, caused dendrites to develop poorly and to have far fewer spines, the knobby dendritic protuberances that form the receiving half of most excitatory synapses. When Zdhhc8 was restored, these abnormalities were prevented in both cultured neurons and in the brains of adult animals, the authors write in a paper published online on 5 October in Nature Neuroscience.

The authors suggest that the chromosomal deletion in their mouse model blocks the post-translational, enzyme-dependent process known as palmitoylation, in which palmitate, a 16-carbon fatty acid, is added to the side chain of an internal cysteine residue in cell membranes. These long hydrophobic chains help to sort and anchor membrane-bound proteins and orient them in relation to the cytosol, a process that is particularly important in the development of synapses. Unlike other protein-sorting mechanisms, palmitoylation is reversible and dynamic, indispensible factors for regulating synaptic plasticity (see El-Husseini and Bredt, 2002; Greaves and Chamberlain, 2007).

Many missing genes
Over the last several years, researchers have amassed a great deal of converging evidence that implicates abnormalities of the chromosome 22q11.2 region in the pathogenesis of schizophrenia. Some of the most suggestive data come from studies of patients with velocardiofacial syndrome, or VCSF, a disorder characterized by cleft palate, cardiac abnormalities, and facial anomalies that arises from a 1.5-3.0 Mbase microdeletion in chromosome 22q11.2. In addition to their physical characteristics, children with VCSF—also known as 22q11 deletion syndrome (22q11DS)—exhibit distinctive emotional and cognitive deficits, and a third of them develop schizophrenia or schizoaffective disorder in adolescence or adulthood, which has stimulated intense interest in 22q11DS among researchers in psychiatric genetics.

About 7 percent of 22q11DS patients carry a 1.5-Mb deletion of a region that contains 27 known genes. Recent studies, including some by the Gogos and Karayiorgou groups, have focused on the functional role of a number of these genes, including catechol-O-methyltransferase (COMT); the myelin-associated gene PIK4CA; the Nogo-66 receptor gene RTN4R; HTF9C, a gene for an RNA binding protein; and proline dehydrogenase (PRODH) (see SRF related news story).

Earlier this year, Gogos’s and Karayiogou’s laboratories published a paper on a mouse model called Df(16)A+/– that was engineered to closely mimic the human 22q11.2 deletion (Stark et al., 2008). A team led by first authors Kimberly Stark and Bin Xu described how the deletion of Dgcr8, a gene that regulates the biogenesis of microRNAs, had widespread effects on gene expression in the hippocampus and prefrontal cortex. That paper also noted behavioral and cognitive deficits, including impairment of prepulse inhibition and fear conditioning and poor performance in spatial-learning tasks, and documented a reduction in dendritic complexity and density of dendritic spines in hippocampal pyramidal neurons (see SRF related news story).

Reversible damage
All but one of the genes affected by 22qDS lie on an orthologous region of mouse chromosome 16 (albeit in a different order), and the deletion in this model covers the same genetic ground as the 1.5 Mbase deletion seen in humans. In the new paper, a group led by first author Jun Mukai concentrated on Zdhhc8, which codes for a putative palmitoyltransferase.

As in the previous work, the researchers again noted a significant reduction in the number of so-called mushroom dendritic spines, a modest but significant reduction in these structures’ length and width, and a reduction in markers associated with pre- and postsynaptic sites.

To tease out the possible role of Zdhhc8 deletion in these effects, the group transfected Df(16)A+/– neurons in culture with a full-length ZDHHC8 expression construct. This manipulation restored the density of dendritic spines and of pre- and postsynaptic markers to wild-type levels, but did not reverse the morphological anomalies in Df(16)A+/– spines, which prompted the authors to propose that dendritic spine size and shape may be governed by Dgcr8 or by other genes deleted in the Df(16)A+/– model.

To further home in on Zdhhc8 deficiency as the possible culprit in the reduction of dendritic spines seen in the Df(16)A+/– mouse, the team examined neurons from homozygous and heterozygous Zdhhc8-knockout mice. They found a dose-dependent reduction in spine density and pre- and postsynaptic markers, and there was no noticeable effect on spine length or width.

In separate experiments, the team documented significant reductions in dendritic complexity in cultured Df(16)A+/– neurons, defined as the number of primary dendrites, the number of their branch points, and their total length. These deficits could also be reversed with ZDHHC8 transfection.

All of these effects seen in vitro on dendritic spine density and complexity, and on pre- and postsynaptic protein markers, were also found in hippocampal neurons from adult Df(16)A+/– and Zdhhc8-knockout mice, though the effects were somewhat smaller than those seen in the experiments in culture, a difference the authors attribute to compensatory developmental mechanisms in the intact animals.

Finally, the researchers showed that PSD95, an important and abundant postsynaptic density protein that has been shown to regulate the number of dendritic spines (Vessey and Karra, 2007), is palmitoylated by Zdhhc8. When this enzymatic process was blocked, normal trafficking of PSD95 to the membrane was disrupted; instead of accumulating in perinuclear regions—a
phenomenon that precedes the clustering of the protein in dendrites, where
it provides scaffolding for postsynaptic ion channels (El-Husseini et al., 2000)—PSD95 was located diffusely in the cytoplasm.

The authors propose that disrupted ZDHHC8-dependent palmitoylation, combined with aberrant miRNA biogenesis, contributes to the behavior and cognitive phenotypes seen in 22q11DS. They go on to suggest that hemi-deletion of the COMT and PRODH genes found in this chromosomal region, and consequent abnormal dopamine neurotransmission, “could further alter network properties, modify the cognitive phenotypes and possibly predispose a subset of 22q11 microdeletion carriers to psychiatric symptoms.”—Peter Farley.

The common theory held until recently regarding the genetic underpinning of neuropsychiatric disorders was based on the “common disease-common variant” model. According to that theory, multiple common alleles in the population contribute small-to-moderate additive or multiplicative effects to the predisposition to neuropsychiatric disorders. With the advances in genetic screening technologies this theory is now being challenged. Recent findings indicate that rare copy number variations (CNVs) may account for a substantial fraction of the overall genetic risk for neuropsychiatric disorders including schizophrenia and autism (Consortium, 2008; Stefansson et al., 2008;
Mefford et al., 2008). The 22q11.2 microdeletion was the most common CNV identified in patients with schizophrenia in a recent large scale study of patients with schizophrenia (Consortium, 2008). The 22q11.2 microdeletion is also the most common microdeletion occurring in humans and up to one third of individuals with 22q11.2 deletion syndrome (22q11.2DS) develop schizophrenia by adulthood. Thus the syndrome serves as an important model from which to learn the path leading from a well defined genetic defect to brain development and eventually to the evolution of schizophrenia.

The current very elegant study by Mukai and colleagues suggests that haploinsufficiency of a single gene from the 22q11.2 deleted region, Zdhhc8, is responsible for the microscopic neural hippocampal abnormalities present in a mouse model of the disease. Remarkably, these abnormalities were prevented with the reintroduction of enzymatically active ZDHHC8 protein. The works of Gogos and his colleagues (Paterlini et al., 2005; Stark et al., 2008) are consistently and brilliantly getting us closer to revealing the complex association between genes from the 22q11.2 region and the neuropsychiatric phenotype. If indeed haploinsuffiency of single genes like Zdhhc8, COMT, or Dgcr8 have a strong effect on abnormal brain development and the eruption of schizophrenia, it conveys an enormous potential for developing novel pathophysiologically based treatments for this refractory disease. Such treatments will target the enzymatic deficit conveyed by the genetic mutation.